The best choice of double burst stimulation pattern for manual evaluation of neuromuscular transmission.
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Journal Article↗
Biomedical subjects
H Tsuda
Publications and source records attributed to H Tsuda.
Changes in gap junction protein (connexin 32) gene expression during rat liver carcinogenesis.
A rat liver gap junction (GJ) cDNA probe that detects mRNA encoding the 32 Kd GJ-protein (connexin 32) was employed to study GJ-protein gene expression in rat liver tumors induced by a single exposure to diethylnitrosamine (DEN) followed by exposure to 2-acetylaminofluorene (AAF)/CCl4/AAF or induced by systemic administration of N-ethyl-N-hydroxyethylnitrosamine (EHEN). All carcinomas generated by these carcinogens showed markedly reduced levels of GJ-protein mRNA. This may indicate that GJ-protein levels and gap-junctional intercellular communication (GJIC) capacity are also severely compromised. Moreover, all hyperplastic nodules also showed a reduced level of GJ-protein mRNA. Taken together with our earlier finding that the liver tumor promoter phenobarbital inhibits GJ-protein gene expression, these results suggest that deranged GJIC is a relatively early event in liver multistage carcinogenesis. A range of other cDNA probes was also used to characterize gene expression in the DEN-induced tumors. Induction of expression was seen for glutathione S-transferase (placental form) (GST-P), gamma-glutamyltranspeptidase (GGT), and c-raf but not for c-Ha-ras or c-myc.
Animals↗
Comparison of enzyme phenotypes in human bladder tumours and experimentally induced hyperplastic and neoplastic lesions of the rat urinary bladder. A combined histochemical and immunohistochemical approach.
The expression of a number of enzymes involved in drug metabolism, membrane function etc. was compared in hyperplastic and neoplastic lesions of the rat bladder and in human bladder tumours. Transitional cell carcinomas (TCC) in both rat and Man were characterized by decreased alkaline phosphatase (ALP) and increased gamma-glutamyl transpeptidase (GGT), beta-glucuronidase (beta-G1), succinate dehydrogenase (SD) and glucose-6-phosphate dehydrogenase (G6PD) activities. In addition, binding for antibodies specific for different cytochrome P-450 species (UT50, PB3a, MC1, MC2) and microsomal epoxide hydrolase (mEHb) was elevated in both murine and human tumours. Comparison of the enzyme phenotype in hyperplastic lesions induced by freeze ulceration or uracil administration with that in preneoplastic papillary or nodular hyperplasia (PNH) and TCC suggested, however, that most of the alteration in enzyme content or activity was non-specific and related to requirements for epithelial cell proliferation. On the other hand, the decreased ALP, and increased GGT and beta-G1 activity appeared more directly related to neoplastic transformation. The results suggested that qualitative differences exist between reactive hyperplasia and preneoplastic or neoplastic lesions in the urinary bladder. The finding of increased cytochrome P-450, in clear contrast to the reduction characteristic of preneoplastic hepatic lesions, may be important with regard to the observed difference in neoplastic transformation between the bladder and liver in response to drug metabolising enzyme inducers.
Alkaline Phosphatase↗
Hepatotoxicity of agents that enhance formation of focal hepatocellular proliferative lesions (putative preneoplastic foci) in a rapid rat liver bioassay.
The histopathology of hepatic toxicity for 58 chemicals previously tested in a rapid rat liver bioassay for demonstrating potential hepatocellular carcinogens and/or tumor promoters was reviewed. Rats received the test diet for 1 week prior to partial hepatectomy and for an additional 5 weeks thereafter at doses near the estimated maximally tolerated dose. These rats served as controls for others receiving initiation by N-nitrosodiethylamine (DEN) and the test diets. Twenty-two of these chemicals were previously found to enhance the formation of glutathione S-transferase, placental form (GST-P)-positive putative preneoplastic hepatocellular foci (promoters) following DEN initiation in this rapid bioassay, whereas 36 chemicals did not. Of the agents that promoted GST-P-positive foci, 14/22 (63.6%) produced toxic hepatocyte lesions while only 4/36 (11.1%) of the nonpromoters did so at the doses used. Biliary toxicity was found for 7/22 (31.8%) of the promoters and 6/36 (16.7%) of the nonpromoters. Only 2/13 (15%) chemicals that inhibited GST-P-positive foci produced hepatic toxicity. Thus, agents that were presumed hepatic tumor promoters characteristically were hepatotoxins while nonpromoters of carcinogenesis were not hepatotoxins in this rapid rat liver bioassay.
Animals↗
Placental form of glutathione S-transferase in normal and diseased human uterine cervical mucosa.
Expression of the human placental form of glutathione S-transferase (GST-pi), an enzyme proposed as a marker for human and experimental neoplasia, was immunohistochemically evaluated in 51 samples of 'normal' and diseased adult human uterine cervix. Five fetal uteri were also studied. GST-pi positivity was detected in 54, 92, 95 and 83% of the 'normal', non-neoplastic, cervical intra-epithelial neoplasia (CIN) and cancer cases respectively. All five fetal uteri and the positive 'normal' adult cases presented cells immunostained for GST-pi throughout the thickness of the mucosa, including the basal layer. Some non-neoplastic conditions like inflammation, repair and metaplasia and some dysplastic and neoplastic lesions showed areas of positively stained cells within an otherwise negative tissue, indicating a phenotypic heterogeneity regarding the enzyme expression. Our results confirm that GST-pi has a fetal character and indicate that it may appear in the adult cervical squamous epithelia under 'normal' or pathological conditions not necessarily linked to the process of carcinogenesis. Therefore it cannot be used as a marker for cervical epithelial neoplasia.
Biomarkers↗
Multicentric independent development of hepatocellular carcinoma revealed by analysis of hepatitis B virus integration pattern.
Two separate nodular lesions of hepatocellular carcinoma (HCC) were surgically removed from a patient with positive serum hepatitis B surface antigen. Southern blot analysis of DNA from the two separate HCC lesions showed different restriction patterns of integrated hepatitis B virus DNA, indicating their independent origins. The two lesions were composed mainly of poorly differentiated HCC; however, well-differentiated HCC was also observed in the periphery of the lesions. This histological finding is consistent with the multistep and independent development of HCC in each site of the liver.
Aged↗
Strain differences in susceptibility to 2-acetylaminofluorene and phenobarbital promotion of rat hepatocarcinogenesis in a medium-term assay system: quantitation of glutathione S-transferase P-positive foci development.
Strain differences in susceptibility to promotion by the liver carcinogens 2-acetylaminofluorene (2-AAF) and phenobarbital (PB) were examined in the medium-term bioassay system initially developed in our laboratory using male F344 rats as the test animal and glutathione S-transferase placental form (GST-P)-positive foci as the lesion end-point. Numbers and areas per cm2 of induced GST-P-positive hepatocellular foci were compared in LEW, F344, NAR, SD, WBN, SHR, Wistar and ODS rats initiated with diethylnitrosamine (DEN) and subjected to partial hepatectomy during subsequent administration of 2-AAF or PB. LEW, SD, WBN, and F344 rats were most susceptible to hepatopromotion by both compounds, with a hundred fold increase in lesion area being observed for 2-AAF in the LEW case. NAR and SHR strains demonstrated an intermediate response, while Wistar and, in particular, the related ODS rats demonstrated very low susceptibilities. The obvious strain differences could be expressed in terms of comparative indices of promoting effects of 2-AAF and PB as well as DEN itself regarding each of the 8 strains tested. The use of F344 rats for the bioassay model was validated by the relatively high sensitivity to both DEN and 2-AAF initiation as well as second-stage promotion stimulus exhibited.
2-Acetylaminofluorene↗
Synergistic effects of low-dose hepatocarcinogens in induction of glutathione S-transferase P-positive foci in the rat liver.
The effects of combined administration of hepatocarcinogens at low doses on the development of glutathione S-transferase P-form (GST-P)-positive foci of rat liver were examined utilizing a bioassay model which consists of a single injection of diethylnitrosamine (DEN, 200 mg/kg, ip), two-thirds partial hepatectomy at week 3 and a 6-week administration of test compounds. The chemicals used, 2-acetylaminofluorene (2-AAF), 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB), phenobarbital (PB), thioacetamide (TAA), N-ethyl-N-hydroxyethylnitrosamine (EHEN), benzo[a]pyrene (B[a]P), carbazole, and alpha-hexachlorocyclohexane (alpha-HCH) were incorporated in the diet, except for EHEN which was dissolved in the drinking water, at levels of 1/6 of the doses usually used. The combinations were: I) 2-AAF, 3'-Me-DAB, PB, TAA, EHEN and B[a]P, II) 2-AAF, 3'-Me-DAB and PB, III) TAA, EHEN and B[a]P, IV) 2-AAF, 3'-Me-DAB, carbazole, TAA, EHEN and alpha-HCH, V) 2-AAF, 3'-Me-DAB and carbazole, and VI) TAA, EHEN and alpha-HCH. All combinations, except for II, caused an increase in the area of the foci as evaluated by the ratios of areas in the combined administration groups to the sum totals of 3 or 6 individual data: I) 1.75, II) 0.81, III) 2.01, IV) 3.62, V) 1.34 and VI) 2.91. The non-synergistic effect in combination II might be related to PB induction of hepatic microsomal enzymes leading to enhanced enzymatical detoxification of 2-AAF and 3'-Me-DAB. The present results indicate that exposure to several chemicals of similar organotropism, even at doses lower than the apparent carcinogenic levels, might be critical to the carcinogenic process.
2-Acetylaminofluorene↗
Strain differences in susceptibility to 2-acetylaminofluorene and phenobarbital promotion of hepatocarcinogenesis: immunohistochemical analysis of cytochrome P-450 isozyme induction by 2-acetylaminofluorene and phenobarbital.
Strain differences in the expression of cytochrome P-450 isoenzymes (P-450s) during enhancement of hepatocarcinogenesis by 2-acetylaminofluorene (2-AAF) and phenobarbital (PB) were investigated immunohistochemically using monoclonal antibodies against phenobarbital (PB) (APF3) or 3-methylcholanthrene (3-MC) (APH8) inducible P-450s. LEW, SD, WBN, F344, SHR, NAR, Wistar and ODS rats were studied, the first five strains proving to be less susceptible to 2-AAF induction of APH8 while responding strongly to the promoting influence of this chemical, as reported previously. The other three strains, NAR, Wistar and ODS, demonstrated greater inducibility, this correlating with an observed resistance to promotion by 2-AAF. PB administration was not associated with any strain difference in APF3 cytochrome P-450 inducibility except in the ODS rat, in which its effects were minimal. The results provide direct evidence that differential expression of cytochrome P-450 species plays a major role in determining responsiveness to hepatocarcinogenesis-promoting agents such as 2-AAF.
2-Acetylaminofluorene↗
Low incidence of point mutation of c-Ki-ras and N-ras oncogenes in human hepatocellular carcinoma.
We examined the incidence of point mutation in codons 12, 13 and 61 of c-Ki-ras and N-ras genes in human hepatocellular carcinoma (HCC) using the polymerase chain reaction and oligonucleotide hybridization techniques. Among 34 tissues specimens surgically resected from 30 patients and 5 cell lines of human HCC, only two had ras point mutations; in one case, codon 12 of c-Ki-ras was altered from GGT, coding glycine, to GTT, coding valine; in the other case, codon 61 of N-ras was altered from CAA, coding glutamine, to AAA, coding lysine. Thus, point-mutational activation of ras oncogenes is an uncommon event in human HCC.
Carcinoma, Hepatocellular↗
Advantages and limitations of stereological estimation of placental glutathione S-transferase-positive rat liver cell foci by computerized three-dimensional reconstruction.
The applicability to a medium-term bioassay for liver carcinogens of mathematical formulae for the calculation of numbers of foci per volume was examined in F344 rats. Two weeks after initiation with diethylnitrosamine, animals were given test compounds for 6 weeks, partial hepatectomy being performed at week 3. At week 8, the rats were killed, the livers removed and stained immunohistochemically for assessment of glutathione S-transferase P form (GST-P)-positive foci development. Numbers and areas of lesions were measured two-dimensionally using a color image analyzer, and the Enzmann and Campbell formulae for estimation of number and volume per cm3 were applied to the results. In addition, three-dimensional reconstruction of individual foci was performed using up to 150 GST-P stained foci, with the aid of a computerized graphic system. Both two- and three-dimensionally expressed quantitative results were found to adequately demonstrate the modifying potential of test chemicals on hepatocarcinogenesis. The three-dimensional approach was only more accurate if most of the foci were small and the liver was enlarged by compound treatment. Stereological reconstruction revealed that the shape of GST-P-positive foci, especially if relatively large, is not always spherical but that many demonstrate irregular branching forms, so that the assumptions behind stereological estimation are not met. The results therefore show that care must be taken in applying mathematical formulae for the calculation of three-dimensional data.
Animals↗
Quantitative autoradiographic study of benzodiazepine receptors: comparative study between normal ddY mouse and El mouse.
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Animals↗
Medium-term bioassay system for detection of carcinogens and modifiers of hepatocarcinogenesis utilizing the GST-P positive liver cell focus as an endpoint marker.
We have developed a medium-term bioassay system of 8 weeks duration utilizing male Fischer 344 (F344) rats for detection of liver carcinogens and modifiers of hepatocarcinogenesis. The system consists of a single intraperitoneal injection of diethylnitrosamine (DEN, 200 mg/kg), 6-weeks-administration of test chemical beginning 2 weeks after the DEN injection, and 2/3 partial hepatectomy (PH) performed at week 3. Carcinogenic potency of test chemicals is predicted based on the results of quantitative analyses of immunohistochemically-demonstrated glutathione S-transferase placental form (GST-P) positive liver cell foci. At present, a total of 140 chemicals have been tested using this system, and the findings show a good correlation with reported carcinogenic activities in long-term tests. Furthermore, the reliability of the system has been extensively examined: the results from the medium-term bioassay were compared with those from long-term experiments using the same doses of selected chemicals; the data from presently-used 2-dimensional analysis were compared with calculated values utilizing mathematical formulae for three-dimensional analysis: conformity of phenotypic expression of enzymes in preneoplastic lesions was examined in relation to their growth activity. In conclusion, although the results with non-hepatocarcinogens were less than satisfactory, the present experimental protocol, which requires far fewer animals and shorter duration than a long-term carcinogenicity test, appears of advantage for rapid screening of the large number of environmental chemicals which may possess hazard potential for induction of liver cancer in man.
Animals↗
Lack of modifying effects of 2,2'-[(4-aminophenyl) imino] bisethanol sulfate on induction of preneoplastic gamma-glutamyl transpeptidase-positive foci in a medium-term bioassay system using F344 rats.
The modifying effects of 2,2'-[(4-aminophenyl) imino] bisethanol sulfate (4APE) on liver carcinogenesis were investigated in male F344/DuCrj rats initially treated with N-nitrosodiethylamine (DEN). Two weeks after a single dose of DEN (200 mg/kg, intraperitoneally), rats were given 4APE at dietary levels of 1,000, 330 and 110 parts per million (ppm), or 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) at 600 ppm as a positive control for 6 weeks. At week 3 following DEN administration, all animals were subjected to partial hepatectomy. No adverse effects on survival and body weight were seen in rats treated with 4APE, even at the highest dietary levels. Marked growth retardation and significant increase of relative liver weight were found in animals treated with the known hepatocarcinogen 3'-Me-DAB, which demonstrated marked promoting activity as evidenced by significantly increased values for gamma-glutamyl transpeptidase (gamma-GT) positive foci as compared with the controls given DEN alone. In contrast, 4APE did not significantly increase the level of gamma-GT positive foci over that induced by DEN initiation alone. From these results, it is concluded that 4APE does not possess promoting activity for liver carcinogenesis.
Animals↗
Rapid bioassay methods for carcinogens and modifiers of hepatocarcinogenesis.
It is very important to detect environmental carcinogens in a short period. For this purpose, a rapid bioassay system based on two-step hepatocarcinogenesis has been developed in our laboratory. Rats were initially given a single dose (200 mg/kg) of diethylnitrosamine (DEN) i.p. and, starting 2 weeks later, were treated with test compounds for 6 weeks and then sacrificed, all rats being subjected to a two thirds partial hepatectomy at week 3. Carcinogenic potential was scored by comparing the glutathione S-transferase placental form-positive foci in the liver with those of the corresponding control. More than 90% of hepatocarcinogens showed positivity, and none of the compounds reported as noncarcinogenic demonstrated positivity. Furthermore, this system also detected inhibitory effects. In order to detect nonhepatocarcinogens, other appropriate systems also have been developed, for example, using methylnitrosourea or other multispectrum carcinogens. These rapid bioassay systems are particularly useful for the screening of environmental carcinogens.
Animals↗
Mode of action of erythropoietin (Epo) in an Epo-dependent murine cell line. I. Involvement of adenosine 3',5'-cyclic monophosphate not as a second messenger but as a regulator of cell growth.
A subclone of the interleukin 3 (IL-3)-dependent murine cell line DA-1 was recently established, and its growth found to be dependent on erythropoietin (Epo) as well as on IL-3. This subclone, named DA-1ER, has been used to study the mechanisms of action of these two growth factors, though most especially of Epo. In the present study, the involvement of adenosine 3',5'-cyclic monophosphate (cAMP) in the growth of DA-1ER cells was analyzed. In initial experiments, cAMP levels in cells stimulated with either recombinant Epo or recombinant IL-3 were monitored. The results showed that no significant changes in cellular cAMP levels had occurred. The cAMP-enhancing agents, N6-2'-o-dibutyryl-adenosine 3',5'-cyclic monophosphate (dbcAMP), 3-isobutyl-1-methyl-xanthine (IBMX), and cholera toxin (CT) were then applied to DA-1ER cells to see whether they could induce cell growth, but none of them was effective. The data indicated that cAMP was not the so-called second messenger for Epo or IL-3. The regulatory effects of cAMP on Epo- and IL-3-stimulated cell growth were next examined. It was found that whereas Epo-stimulated growth was markedly inhibited by cAMP-enhancing agents, IL-3-stimulated growth was relatively resistant and inhibited only by high doses of these agents. These data suggested that cAMP could play different roles in the same cells, depending on which growth factors were applied, and that it finely regulated proliferation and differentiation of multireceptor-bearing hematopoietic precursor cells.
1-Methyl-3-isobutylxanthine↗
Combination therapy of glycyrrhizin withdrawal and human fibroblast interferon for chronic hepatitis B.
In ten carriers positive for chronic hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and DNA polymerase, the authors investigated the efficacy of the combination therapy consisting of glycyrrhizin withdrawal and human fibroblast interferon (locally produced). Glycyrrhizin was given for four weeks and was stopped without tapering off the dose. Human fibroblast interferon was given continuously. Thirty-six weeks after the end of this treatment, three of the ten patients were HBeAg negative but not anti-HBe positive, and in one of these three DNA polymerase became undetectable. Another patient showed a loss of DNA polymerase with HBeAg. Transaminase levels decreased in nine of the patients. Glycyrrhizin appeared to act as an antiviral agent in four patients and had a corticoid-like effect in three. DNA polymerase decreased remarkably after interferon administration, and serum transaminase levels increased. No side effects were reported in patients receiving glycyrrhizin. In contrast, almost all patients receiving human fibroblast interferon had influenza-like symptoms, which, although initially severe, decreased with subsequent injections of interferon. Thus this combination therapy seems safe and effective.
Adult↗
[Study of the clinical background and therapeutic method in patients with vulvar cancer].
Forty-two cases of vulvar cancer were reviewed with regard to clinical condition and therapeutic method, especially the efficacy of local administration of BRM. By histological examination, koilocytosis was found in 45%. Furthermore, there was 90% positive koilocytosis rate in immunohistological study using anti-human papilloma virus antibody and in situ hybridization method by HPV DNA probe (type 6, 11, 16, 18). The HPA was mostly type 16. Therefore, vulvar cancer was considered to occur in close relation with HPV type 16. Some 12% of vulvar cancer was complicated with other gynecologic cancer. In these cases, NK activity was low. Thus, cancer may be considered to occur in association with low immunological status. With regard to the therapeutic method, surgery and radiotherapy were usually used after chemotherapy in our institute. At stage 3-4, combination therapy using surgery and radiotherapy were found to be most effective. In regard to local immunotherapy using OK-432 and LAK cell and IL-2, local administration of OK-432 was effective. Therefore, surgery and radiotherapy with local immunotherapy after chemotherapy were considered to be the most effective therapeutic method for vulvar cancer.
Aged↗